Clot formation in plasma is initiated by the serial enzymatic activation of clotting factors, which results in the appearance of small traces of thrombin. Hurlet-Birk Jensen A., et al. Factor V and VIII Activation "In Vivo" during Bleeding. Evidence of Thrombin Formation at the Early Stage of Hemostatasis. Path. Biol. 1976; 24, p.6-10. The explosive nature of the coagulation process is a consequence of the position feedback reactions that these thrombin traces exert on the cofactors V. Colman, R W, The Effect of Proteolytic Enzymes on Bovine Factor VI. Kinetics of Activation and Inactivation by Bovine Thrombin, Biochem. 1969; 4: p.1438-1444, Lindhout, M J, Activation of Bovine Factor V by Thrombin and a Protease from Russell's Viper Venom (RVV), Thromb. Haemost. 1979; 42: p.491. Rapaport, S I, et al., Further Evidence That Thrombin Activation of Factor VIII is an Essential Step In Intrinsic Clotting, Scand J. Clin. Lab. Invest. 1965; 17: p.84-88, Biggs, R., et al. Thrombin and the Interaction of Factors VIII and IX, Brit J. Haemat. 1965; 11: p.276-295, Hemker, H C, et al., Reaction Sequence of Blood Coagulation, Nature 1967; 215: p.1201, Osterud, B., et al., Formation of Intrinsic Factor X Activator With Special Reference to the Role of Thrombin, Br. J. Haematol 1971; 21: p.643-660, Hultin, M B, et al., Activation of Factor X by Factors IXa and VIII; A Specific Assay for Factor IXa in the Presence of Thrombin-Activated Factor VIII, Blood 1978; 52: p.928-940, and on platelets, Davey, M G, et al., Actions of Thrombin and Other Proteolytic enzymes on Blood Platelets, Nature 1967; 216: p.857-858. Activated Factors V and VIII:C dramatically boost the performance of Factors Xa and IXa, whereas activated platelets provide, among other things, the negatively charged surface necessary for most coagulation reactions. Bevers, E M, et al., Generation of Prothrombin Converting Activity and the Exposure of Phosphatidylserine at the Outer Surface of Platelets, Eur. J. Bioch. 1982; 122: p.429-436.
According to prior methods Factor VIII:C concentrations, and also Factor VIII:C activation in plasma were measured using coagulation assays, involving VIII:C deficient plasma. Soulier, P., et al., Deficit En Beme Facteur Prothromboplastique plasmatique, Rapports Entre le PTA et le Facteur Hageman, Thrombin. Diathes. Haemorrh. 1958; 2: p.1, Rappaport, S I, et al., A Simple Specific One-Stage Assay for Plasma Thromboplastin Antecedent Activity, J. Lab. Clin. Med. 1961; 57: p.771, Hardisty, R M et al., A One-Stage Factor VIII (Antihemophilic Globulin) Assay and Its Uses on Venous and Capillary Plasma, Thrombos, Diathes, Haemorrh. 1962; 7: p.215, Veltkamp, J J, et al., Detection of the Carrier State in Hereditary Coagulation Disorders, Thrombos, Diathes. Haemorrh. 1968; 19: p.279-303 and 403-422, Suomela, H, et al. The Activation of Factor X Evaluated by Using Synthetic Substrates, Thromb. Res. 1977; 1: p.267-281, Van Dieijen, G, et al., The Role of Phospholipid and Factor VIIIa in the Activation of Bovine Factor X, J. Biol. Chem. 1981; 256: p.3433-3442. The occurrence of various feedback reactions, however, made it impossible to relate, in a quantitatively reliable way, these values to the amounts of activated VIII:Ca. The advent of a chromogenic substrate for Factor Xa, and the recognition that a property of activated Factor VIII:Ca is accelerated activation of Factor X by IXa, phospholipids and calcium ions made a more direct method of determining functional Factor VIII:Ca conceivable. Suomela, H, et al. Supra, Van Dieijen, et al., The Role of Phospholipid and Factor VIIIa in the Activation of Bovine Factor X, J. Biol. Chem. 1981; 256: p.3433-3442. Still, feedback reactions of Factor Xa on Factor VIII:C, and the inactivation of Factor Xa by the antithrombin III and .alpha.1-antitrypsin present in plasma, threatened to seriously hamper the feasibility of such a Factor VIII:Ca assay in plasma. Vehar, G A, et al., Preparation and Properties of Bovine Factor VIII (Antihemophilic Factor), Biochemistry 1980; 19: p.401-410, Hultin, M B, Role of Human Factor VIII in Factor X Activation, J. Clin. Invest. 1982; 69: p.950-955, Lollar, P, et al., Activation of Porcine Factor VIII:C by Thrombin and Factor Xa, Biochemistry 1985; 24: p.8056-8064, Neuenschwanter, P, et al., A Comparison of Phospholipid and Platelets in the Activation of Human Factor VIII by Thrombin and Factor Xa, and in the Activation of Factor X, Blood 1988; 72: p.1761-1770, and Factor X, Jesty, J., et al., The Mechanism of Activation of Factor X, J. Biol. Chem. 1974; 249: p.5614-5622. On the basis of a method by Pieters et al., In Situ Generated Thrombin is the Only Enzyme that Effectively Activates Factor VIII:C and Factor V in Plasma, Blood, in press (hereby incorporated by reference), we developed a scheme for measuring Factor VIII:Ca in plasma, that circumvents these difficulties.
The classical method for the determination of Factor VIII:Ca activity in plasma estimates the activity of the clotting factor in a plasma sample from the amount of time by which it shortens the prolonged clotting time of a plasma cogenitally deficient in that factor, as compared to a normal plasma. As there is no solid theoretical basis that underlies the relationship between the coagulation time and the percentage of coagulation factor activity, these assays are bioassays in the sense that the activities have to be read from a standard curve, and that the values of these activities are only valid with respect to the method by which they are measured. Consequently, a need exists for a more quantitative method to measure Factor VIII:Ca.